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Hierarchical clustering and network analysis for the 16 PAHs. (A) Hierarchical clustering of the DEGs of the 16 PAHs compared to the control samples. Genes with elevated expression are in yellow and genes with reduced expression are in blue. Genes were ranked by the coefficient of variation and only the top 500 genes are included in the heatmap. (B) Network inferred using the Context Likelihood of Relatedness (CLR) program that links the 16 PAHs based on coordinated transcription of genes as they respond to each PAH. The nodes in the network are specific PAHs, with the colors representing the developmental toxicity bins they belong to. The two controls (in black) are included in the figure. The connectors are similarity of transcriptome response to those PAHs. The thicker the connector, the more similar the response of the PAHs. To produce a more relevant network, only the top 500 genes based on coefficient of variation (CV) was used. Abbreviations: Cont = Control.
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Polycyclic Aromatic Hydrocarbons (PAHs) are diverse environmental pollutants associated with adverse human health effects. Many studies focus on the carcinogenic effects of a limited number of PAHs and there is an increasing need to understand mechanisms of developmental toxicity of more varied yet environmentally relevant PAHs. A previous study ch...
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... order to identify PAHs with similar and unique transcriptomic responses, we performed hierarchical clustering of PAHs using expression profiles for the 500 genes with the highest coefficient of variation (CV) across the 18 conditions (16 PAH treatments and 2 controls) ( Figure 3A). For a list of the top 5000 genes with the highest CV across the 18 treatments, see Table S3. ...
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... again used only the top 500 genes based on the CV to ensure a more relevant network as basal level responses of genes with a low CV are not present to dilute the effect of the most responsive genes. This analysis also clustered the 16 PAHs into the same two broad clusters ( Figure 3B). Interestingly, there were PAH pairs with similar developmental toxicity profiles that binned together in our phenotypic screening heatmap (Figure 2) yet had less related transcriptomic The larger cluster (Cluster A) ( Figure 3A,B) showed more varied Cyp1a localization patterns and modest or no phenotypic response to PAH exposure. ...
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... analysis also clustered the 16 PAHs into the same two broad clusters ( Figure 3B). Interestingly, there were PAH pairs with similar developmental toxicity profiles that binned together in our phenotypic screening heatmap (Figure 2) yet had less related transcriptomic The larger cluster (Cluster A) ( Figure 3A,B) showed more varied Cyp1a localization patterns and modest or no phenotypic response to PAH exposure. Many of these PAHs, specifically 1,5-DMN, 2-MN, 3-NF, acenaphthene, anthracene, and phenanthrene also showed zero or very few DEGs. ...
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... of these PAHs, specifically 1,5-DMN, 2-MN, 3-NF, acenaphthene, anthracene, and phenanthrene also showed zero or very few DEGs. All of these PAHs except 3-NF are from the less developmentally toxic Bins 6-8, and share connectors with the control samples ( Figure 3B). This indicates the similarity of the transcriptomic responses of these PAHs to the control samples at 48 hpf. ...
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... smaller cluster (Cluster B) ( Figure 3A,B) consisted of retene, BkF, BjF, DB(a,i)P, DB(a,i)P, and BbF, all PAHs belonging to the more developmentally toxic bins 1-5. These six PAHs localized Cyp1a protein expression in the vasculature. ...
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... were other similarities between Cluster B PAHs: BjF and BkF (Bin 2) both showed an unusual caudal fin phenotype [45], and are structurally similar five-ring PAHs with only one of the rings positioned differently [69]. These PAHs are also adjacent to each other in our transcriptomic response dendrogram ( Figure 3A), showing that transcriptomic and phenotypic responses of these PAHs are tightly correlated. The other two PAHs in Bin 2 (carbazole and 3-NF) are less structurally similar and did not cluster tightly with each other or with BkF and BjF, further suggesting different molecular signaling events. ...
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... other two PAHs in Bin 2 (carbazole and 3-NF) are less structurally similar and did not cluster tightly with each other or with BkF and BjF, further suggesting different molecular signaling events. DB(a,h)P (Bin 4) and BbF (Bin 5) had the most similar gene expression changes depicted by their close proximity in the dendrogram (Figure 3A), and the thickness of the connector between the two PAHs ( Figure 3B). Both PAHs despite being in different developmental toxicity bins (Figure 1), produced aberrant behavioral phenotypes with no malformations. ...
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... other two PAHs in Bin 2 (carbazole and 3-NF) are less structurally similar and did not cluster tightly with each other or with BkF and BjF, further suggesting different molecular signaling events. DB(a,h)P (Bin 4) and BbF (Bin 5) had the most similar gene expression changes depicted by their close proximity in the dendrogram (Figure 3A), and the thickness of the connector between the two PAHs ( Figure 3B). Both PAHs despite being in different developmental toxicity bins (Figure 1), produced aberrant behavioral phenotypes with no malformations. ...
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... there were PAH pairs with similar developmental toxicity profiles that binned together in our phenotypic screening heatmap (Figure 2) yet had less related transcriptomic responses (and vice versa). Retene and 4h-CPdefP are highly disparate in the dendrogram in Figure 3A and clustered separately in our network analysis ( Figure 3B) but had similar developmental toxicity profiles (high mortality with morphological and behavioral endpoints in common). The same was observed for the Bin 4 PAHs DB(a,h)P and 9-MA which caused behavioral effects, but were far apart in the dendrogram and the network (Figure 3A,B). ...
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... there were PAH pairs with similar developmental toxicity profiles that binned together in our phenotypic screening heatmap (Figure 2) yet had less related transcriptomic responses (and vice versa). Retene and 4h-CPdefP are highly disparate in the dendrogram in Figure 3A and clustered separately in our network analysis ( Figure 3B) but had similar developmental toxicity profiles (high mortality with morphological and behavioral endpoints in common). The same was observed for the Bin 4 PAHs DB(a,h)P and 9-MA which caused behavioral effects, but were far apart in the dendrogram and the network (Figure 3A,B). ...
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... and 4h-CPdefP are highly disparate in the dendrogram in Figure 3A and clustered separately in our network analysis ( Figure 3B) but had similar developmental toxicity profiles (high mortality with morphological and behavioral endpoints in common). The same was observed for the Bin 4 PAHs DB(a,h)P and 9-MA which caused behavioral effects, but were far apart in the dendrogram and the network (Figure 3A,B). Cyp1a expression patterns were also different between these pairs of PAHs. ...
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... BaP shows mixed effects at the larval stage, although additional deficits such as altered locomotor activity, startle responses and memory performance have been noted in later stages of development. Comparatively, there is little data on the neurobehavioral toxicity of FA, with existing studies limited to embryo/larval testing (Geier et al., 2018;Shankar et al., 2019). A number of studies have tested the neurobehavioral toxicity of CD in zebrafish, although this too is generally limited to embryo/larval testing LeFauve and Connaughton, 2017;Liao et al., 2021;Shankar et al., 2021;Xu et al., 2022). ...
... This includes low molecular weight (LMW) PAH compounds such as phenanthrene (Phen), naphthalene (Nap), acenaphthene (Ace), anthracene (Anth), acenaphthylene (Acy), fluorene (Flu), and high molecular weight (HMW) PAH compounds such as chrysene (Chr), fluoranthene (Flut), pyrene (Pyr), benzo [b] (Tongo et al. 2017;Wang et al. 2019a;Patel et al. 2020). This is a major environmental and societal concern as they are linked to developmental toxicity, mutagenicity, neurotoxic, oxidative stress, and hormonal disruption in various toxicological studies (Shankar et al. 2019;Reguera et al. 2018). PAHs are among the most severe organic pollutants due to their ubiquity in the natural environment and cause many detrimental impacts on organisms. ...
Polycyclic aromatic hydrocarbons (PAHs) are organic chemicals that can induce oxidative stress, genotoxicity, immunotoxicity, endocrine disruption, and developmental toxicity and are carcinogenic. Marine benthic macroinvertebrates are used as biomarkers for elucidating the level of environmental pollution due to their sedentary nature and ability to accumulate toxic compounds over an extended period. Antioxidant defence systems in macroinvertebrates protect cells from reactive oxygen species formed during oxidative stress, and they also counteract the effect of the pollutants through various physiological adaptations and differential expression of specific enzymes. A literature review on molecular studies on various marine benthic macroinvertebrates phyla was evaluated to understand their response to different PAH exposures. Literature shows that genomic tools can define toxicant-specific gene transcriptome variations, which can be utilized to identify the principal pathways that are affected. The review addresses analytical methods, similarities, and differences in antioxidant enzymes and the expression of various genes studied. The comprehensive analysis of literature reveals that several studies have explored the responses of organisms to PAH pollution; this included genes such as CYP450s, GST, SOD, GPx, CAT, and HSPs. Numerous studies have consistently demonstrated notable up-regulations in these genes, establishing their characterization as PAH-sensitive genes, highlighting the critical role played by them for cellular defence and detoxification mechanisms. PAHs can affect organisms depending on exposure time, kind, matrix, and pollutant dose. Benthic macroinvertebrates are robust bioindicators for PAH assessments; thus, environmental risk assessments need a standardized quality and assurance methodology for contamination exposures and biomarker interpretation.
... Systems where RNA-seq has been applied include clonal cell populations in monoculture (Landry et al., 2013), complex microbiomes (Carvalhais et al., 2012;Bashiardes et al., 2016), 3D human tissue models (Chang et al., 2021), and whole organisms including zebrafish (Danio rerio), a model vertebrate organism (Hu et al., 2019). One specific area where RNA-seq has been used is the response of zebrafish to a variety of specific chemicals or other kinds of stresses (Zheng et al., 2018;Shankar et al., 2019;Huang et al., 2020;Dasgupta et al., 2022). Application of RNA-seq has become a common -omics tool and recently more advanced technologies have been developed and applied to zebrafish and other systems. ...
... Application of RNA-seq has become a common -omics tool and recently more advanced technologies have been developed and applied to zebrafish and other systems. These include modifications of RNA sequencing designed to answer specific questions such as Prime-Seq, Decode-Seq, Lasy-Seq (Kamitani et al., 2019;Li et al., 2020;Janjic et al., 2022) singlecell RNA-seq, where specific transcriptomics signatures can be determined from sub-populations of cells (Bageritz and Raddi, 2019;Jiang et al., 2021;Metikala et al., 2021;Tatarakis et al., 2021), sequencing of specific RNA types such as microRNAs (Zayed et al., 2019) and combination of RNA-seq data sets to carry out metanalyses or to determine how genes are coexpressed across conditions (Shankar et al., 2019;De Toma et al., 2021;Shankar et al., 2021). ...
Introduction: The application of RNA-sequencing has led to numerous breakthroughs related to investigating gene expression levels in complex biological systems. Among these are knowledge of how organisms, such as the vertebrate model organism zebrafish ( Danio rerio ), respond to toxicant exposure. Recently, the development of 3′ RNA-seq has allowed for the determination of gene expression levels with a fraction of the required reads compared to standard RNA-seq. While 3′ RNA-seq has many advantages, a comparison to standard RNA-seq has not been performed in the context of whole organism toxicity and sparse data.
Methods and results: Here, we examined samples from zebrafish exposed to perfluorobutane sulfonamide (FBSA) with either 3′ or standard RNA-seq to determine the advantages of each with regards to the identification of functionally enriched pathways. We found that 3′ and standard RNA-seq showed specific advantages when focusing on annotated or unannotated regions of the genome. We also found that standard RNA-seq identified more differentially expressed genes (DEGs), but that this advantage disappeared under conditions of sparse data. We also found that standard RNA-seq had a significant advantage in identifying functionally enriched pathways via analysis of DEG lists but that this advantage was minimal when identifying pathways via gene set enrichment analysis of all genes.
Conclusions: These results show that each approach has experimental conditions where they may be advantageous. Our observations can help guide others in the choice of 3′ RNA-seq vs standard RNA sequencing to query gene expression levels in a range of biological systems.
... It then breaks free from its chaperones and enters the nucleus where it canonically partners with AHR nuclear translocator (ARNT) to bind xenobiotic response elements within the genome. This induces the transcription of numerous genes involved in the xenobiotic response including p450s, wfkkn1n, foxq1a, and nrf2 [18][19][20][21][22]. ...
... The transcriptomic data generated from zebrafish larval exposures to PSD extracts were new to this study while that of the oxygenated PAH (OPAH) and parent PAH compounds have previously been published [21,28]. A summary table of the exposure scenarios and techniques to generate the RNA-Seq data is included in Supplementary Table S2. ...
... The quality was determined with an Agilent Bioanalyzer 2100. We confirmed a RIN score above 9 in each sample before sending the total RNA samples to The University of Oregon Genomics Core, where RNA was poly-A selected via the Dynabead mRNA Purification Kit (Invitrogen), library prepped with the ScriptSec v2 Kit and ScriptSeq index primers, and 50 bp paired-end sequenced with an Illumina HISEQ 2000 sequencer This study also utilized the transcriptomic data measured from larval exposures to the parent PAHs retene (Ret), benzo(k)fluoranthene (BkF), and benzo(b)fluoranthene (BbF) originally generated and published by Shankar et al. [21]. We extracted the raw fastqs of these exposures from the GEO database, ascension number GSE171944. ...
Passive sampling device (PSD) extracts paired with developmental toxicity assays in Danio Rerio (zebrafish) are excellent sensors for whole mixture toxicity associated with the bioavailable non-polar organics at environmental sites. We expand this concept by incorporating RNA-Seq in 48-h post fertilization zebrafish statically exposed to PSD extracts from two Portland Harbor Superfund Site locations: river mile 6.5W (RM 6.5W) and river mile 7W (RM 7W). RM 6.5W contained higher concentrations of polycyclic aromatic hydrocarbons (PAHs), but the diagnostic ratios of both extracts indicated similar PAH sourcing and composition. Developmental screens determined RM 6.5W to be more toxic with the most sensitive endpoint being a “wavy” notochord malformation. Differential gene expression from exposure to both extracts was largely parallel, although more pronounced for RM 6.5W. When compared to the gene expression associated with individual chemical exposures, PSD extracts produced some gene signatures parallel to PAHs but were more closely matched by oxygenated-PAHs. Additionally, differential expression, reminiscent of the wavy notochord phenotype, was not accounted for by either class of chemical, indicating the potential of other contaminants driving mixture toxicity. These techniques offer a compelling method for non-targeted hazard characterization of whole mixtures in an in vivo vertebrate system without requiring complete chemical characterization.
... The toxic effects of petroleum hydrocarbon compounds on fishes and other aquatic organisms have been reported in previous literatures (Doering et al., 2018;Franco and Lavado, 2019;Mahugija and Njale, 2018). Recent studies have highlighted the neurodevelopmental, reproductive toxicities as well as genetic damage, alteration in feeding and locomotive behavior in aquatic organisms Geier et al., 2018;Incardona and Scholz, 2018;Rajendran et al., 2022;Shankar et al., 2019;Shen and Zuo, 2020;Singh et al., 2021;Yamaguchi et al., 2020;Zhang et al., 2022). PAHs has been shown to Cause cardiac deformities in fish (Incardona, 2017;Incardona and Scholz, 2016). ...
Marine oil spills emanating from wells, pipelines, freighters, tankers, and storage facilities draw public attention and necessitate quick and environmentally friendly response measures. It is sometimes feasible to contain the oil with booms and collect it with skimmers or burn it, but this is impracticable in many circumstances, and all that can be done without causing further environmental damage is adopting natural attenuation, particularly through microbial biodegradation. Biodegradation can be aided by carefully supplying biologically accessible nitrogen and phosphorus to alleviate some of the microbial growth constraints at the shoreline. This review discussed the characteristics of oil spills, origin, ecotoxicology, health impact of marine oils spills, and responses, including the variety of remedies and responses to oil spills using biological techniques. The different bioremediation and bio-dispersant treatment technologies are then described, with a focus on the use of green surfactants and their advances, benefits/drawbacks. These technologies were thoroughly explained, with a timeline of research and recent studies. Finally, the hurdles that persist as a result of spills are explored, as well as the measures that must be taken and the potential for the development of existing treatment technologies, all of which must be linked to the application of integrated procedures.
... A quantitative structure activity relationship (QSAR) model called conditional toxicity value (CTV) was also used for chemicals lacking toxicity values [67]. Toxicity testing in zebrafish was conducted for the 63 PAHs that samples were analyzed for and benchmark concentrations values were calculated [68]. International Agency for Research on Cancer (IARC) cancer classification and different sources of potency including relative potency factor (RPF) were also incorporated [69]. ...
There is a growing need to establish alternative approaches for mixture safety assessment of polycyclic aromatic hydrocarbons (PAHs). Due to limitations with current component-based approaches, and the lack of established methods for using whole mixtures, a promising alternative is to use sufficiently similar mixtures; although, an established framework is lacking. In this study, several approaches are explored to form sufficiently similar mixtures. Multiple data streams including environmental concentrations and empirically and predicted toxicity data for cancer and non-cancer endpoints were used to prioritize chemical components for mixture formations. Air samplers were analyzed for unsubstituted and alkylated PAHs. A synthetic mixture of identified PAHs was created (Creosote-Fire Mix). Existing toxicity values and chemical concentrations were incorporated to identify hazardous components in the Creosote-Fire Mix. Sufficiently similar mixtures of the Creosote-Fire Mix were formed based on (1) relative abundance; (2) toxicity values; and (3) a combination approach incorporating toxicity and abundance. Hazard characterization of these mixtures was performed using high-throughput screening in primary normal human bronchial epithelium (NHBE) and zebrafish. Differences in chemical composition and potency were observed between mixture formation approaches. The toxicity-based approach (Tox Mix) was the most potent mixture in both models. The combination approach (Weighted-Tox Mix) was determined to be the ideal approach due its ability to prioritize chemicals with high exposure and hazard potential.
... Zebrafish have been utilized to identify complex chemicaltranscriptome interactions and identify differentially expressed genes (DEGs) important to overall toxicity response (Huang et al., 2014;Huang et al., 2015;Souza et al., 2015;Shankar et al., 2019). One major approach is through RNA sequencing, which provides an unbiased snapshot of all gene expression changes at a given timepoint during development in response to chemical insult (Heijne et al., 2005). ...
... One major approach is through RNA sequencing, which provides an unbiased snapshot of all gene expression changes at a given timepoint during development in response to chemical insult (Heijne et al., 2005). Many transcriptomic studies have revealed important gene expression changes in response to polycyclic aromatic hydrocarbons (PAHs) across model systems (Song et al., 2012;Jayasundara et al., 2015;Brinkmann et al., 2016;Shankar et al., 2019). PAHs are ubiquitous environmental contaminants produced both naturally and anthropogenically through sources like wildfires, crude oil, and cigarette smoke. ...
... While we have a good understanding of what gene expression changes are induced by AHR activation, which of these transcriptional changes are directly associated with toxicity and which are adaptive or benign is not possible to deduce from assessment using one exposure concentration. Retene, a frequently detected PAH in environmental sampling, induces elevated cyp1a expression in developing zebrafish in an AHR2dependent manner and produces malformations similar to those induced by TCDD (Geier et al., 2018b;Shankar et al., 2019). In this study, we leveraged an established developmental zebrafish screening assay and whole-animal transcriptomic analysis to identify concentration-dependent gene expression changes associated with retene teratogenicity. ...
Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants and are associated with human disease. Canonically, many PAHs induce toxicity via activation of the aryl hydrocarbon receptor (AHR) pathway. While the interaction between PAHs and the AHR is well-established, understanding which AHR-regulated transcriptional effects directly result in observable phenotypes and which are adaptive or benign is important to better understand PAH toxicity. Retene is a frequently detected PAH in environmental sampling and has been associated with AHR2-dependent developmental toxicity in zebrafish, though its mechanism of toxicity has not been fully elucidated. To interrogate transcriptional changes causally associated with retene toxicity, we conducted whole-animal RNA sequencing at 48 h post-fertilization after exposure to eight retene concentrations. We aimed to identify the most sensitive transcriptomic responses and to determine whether this approach could uncover gene sets uniquely differentially expressed at concentrations which induce a phenotype. We identified a concentration-response relationship for differential gene expression in both number of differentially expressed genes (DEGs) and magnitude of expression change. Elevated expression of cyp1a at retene concentrations below the threshold for teratogenicity suggested that while cyp1a expression is a sensitive biomarker of AHR activation, it may be too sensitive to serve as a biomarker of teratogenicity. Genes differentially expressed at only non-teratogenic concentrations were enriched for transforming growth factor-β (TGF-β) signaling pathway disruption while DEGs identified at only teratogenic concentrations were significantly enriched for response to xenobiotic stimulus and reduction-oxidation reaction activity. DEGs which spanned both non-teratogenic and teratogenic concentrations showed similar disrupted biological processes to those unique to teratogenic concentrations, indicating these processes were disrupted at low exposure concentrations. Gene co-expression network analysis identified several gene modules, including those associated with PAHs and AHR2 activation. One, Module 7, was strongly enriched for AHR2-associated genes and contained the strongest responses to retene. Benchmark concentration (BMC) of Module seven genes identified a median BMC of 7.5 µM, nearly the highest retene concentration with no associated teratogenicity, supporting the hypothesis that Module seven genes are largely responsible for retene toxicity.
... In the studies that follow the zebrafish, Danio rerio, was used to shed light on developmental toxicity of multiple chemicals and chemical mixtures. The zebrafish provide an excellent in vivo model to understand the genetic consequences to early exposures to chemicals and chemical mixtures (Shankar et al., 2019;M. Zheng et al., 2018). ...
... , the zebrafish provide an excellent in vivo model to understand the genetic consequences to early exposures to chemicals and chemical mixtures and have been used in RNA sequencing based analyses(Barta et al., 2018;Qiu et al., 2019;Shankar et al., 2019;M. Zheng et al., 2018). ...
Exposure to drinking water contaminants has been linked to developmental outcomes in both epidemiological and model organism studies. However, low level mixture effects, on early development has yet to be explored. It is hypothesized that early chemical exposures may increase disease susceptibility later in life. This work aimed to investigate impacts of a variety of chemicals and concentrated on metals arsenic (As), cadmium (Cd), vanadium (V), and lead (Pb) due to their presence in drinking water and known developmental toxicity. To determine the effects of a metal and organic contaminant co-exposure, the ubiquitously used herbicide glyphosate was also explored. The zebrafish (Danio rerio) model was used to elucidate developmental impacts of lowlevel chemical mixture exposure with a focus on mitochondrial function. An in-depth analysis exploring embryonic oxygen consumption rate (eOCR) in response to all iterations of a 5-part chemical mixture of glyphosate, As, Cd, V, and Pb showed that mitochondria are highly sensitive to mixture toxicity, and that pre-exposure to a metal mixture leave the mitochondria more susceptible to acute chemical stress through depleted reserve capacity. Altered mitochondrial function, along with changes in gene expression and histology suggested that early mixture exposure may contribute to the endemic of chronic kidney disease of unknown etiology (CKDu). To investigate underlying molecular mechanisms that may contribute to CKDu susceptibility, RNA seq data from zebrafish embryos exposed to mixtures of As, Cd, V, and Pb, (+/- glyphosate) and glyphosate alone, suggest that exposure to metal and organic mixtures may be altering the extracellular matrix of kidney tissue. This combined with impaired mitochondrial function, could leave individuals more susceptible to kidney injury CKDu progression. To determine phenotypes associated with mixture exposure, changes in behavior after exposure to a large collection of water samples were explored. A cluster analysis of metals found in drinking water samples were coupled to changes in behavior and revealed that concentrations of Pb, Cd, As, Uranium (U) and Nickel (Ni), should be taken into special consideration when determining drinking water standards. These data suggest that impaired mitochondria, as a result of low-level mixture exposure, may function in the early onset of disease, such as CKDu, and further impair organism development.
... Declining cost and widening availability of high-throughput sequencing have made RNA sequencing a popular discovery tool for transcriptomic changes from chemical exposure. Developing zebrafish exposed to Ahr2 activators exhibit several liganddependent transcriptional changes (Goodale et al., 2015;Shankar et al., 2019), and multiple transcriptional factors are predicted to regulate expression of genes associated with Ahr2 activation (Garland et al., 2020;Goodale et al., 2015), emphasizing the intricate crosstalk between pathways upon chemical exposure. ...
... Our group has published multiple transcriptomic studies in developmental zebrafish exposed to various chemicals. The studies identified wfikkn1-whey acidic protein (WAP), follistatin/Kazal, immunoglobulin (IG), Kunitz/bovine pancreatic trypsin inhibitor (BPTI), and netrin (NTR) domain containing 1-as highly induced in 48 h postfertilization (hpf) zebrafish exposed to individual PAHs and TCDD (Garcia et al., 2018b;Goodale et al., 2013Goodale et al., , 2015Shankar et al., 2019). Additionally, a comparison of several of these gene expression studies revealed wfikkn1's high coexpression with cyp1a, suggesting wfikkn1's role in Ahr2 signaling (Shankar et al., 2021). ...
... Previous work has shown similar concentration-dependent expression of cyp1a and other AHR-regulated genes, including ahrra and ahrrb (Evans et al., 2005). Our lab's previous study demonstrated via transcriptomics that, although certain PAHs predominantly activate Ahr2 and consequently induce cyp1a expression, each chemical can elicit unique gene expression signatures (Shankar et al., 2019). We found that despite chemical-specific gene expression changes, wfikkn1 was always induced when cyp1a expression was significantly increased. ...
The aryl hydrocarbon receptor (AHR) is required for vertebrate development and is also activated by exogenous chemicals, including polycyclic aromatic hydrocarbons (PAHs) and TCDD. AHR activation is well-understood, but roles of downstream molecular signaling events are largely unknown. From previous transcriptomics in 48-hours post fertilization (hpf) zebrafish exposed to several PAHs and TCDD, we found wfikkn1 was highly co-expressed with cyp1a (marker for AHR activation). Thus, we hypothesized wfikkn1's role in AHR signaling, and showed that wfikkn1 expression was Ahr2 (zebrafish ortholog of human AHR)-dependent in developing zebrafish exposed to TCDD. To functionally characterize wfikkn1, we made a CRISPR-Cas9 mutant line with a 16-bp deletion in wfikkn1's exon, and exposed wildtype and mutants to DMSO or TCDD. 48-hpf mRNA sequencing revealed over 700 genes that were differentially expressed (p < 0.05, log2FC > 1) between each pair of treatment combinations, suggesting an important role for wfikkn1 in altering both the 48-hpf transcriptome and TCDD-induced expression changes. Mass spectrometry-based proteomics of 48-hpf wildtype and mutants revealed 325 significant differentially expressed proteins. Functional enrichment demonstrated wfikkn1 was involved in skeletal muscle development and played a role in neurological pathways after TCDD exposure. Mutant zebrafish appeared morphologically normal but had significant behavior deficiencies at all life stages, and absence of Wfikkn1 did not significantly alter TCDD-induced behavior effects at all life stages. In conclusion, wfikkn1 did not appear to be significantly involved in TCDD's overt toxicity but is likely a necessary functional member of the AHR signaling cascade.
... Fish exposed to certain PAHs are known to induce cyp1a (Nebert and Gonzalez, 1987;Shankar et al., 2019). Exposure to retene resulted in upregulation of cyp1a alongside cytosolic sulfotransferase (sult1), carbonyl reductase (cbr1l) and UDP-glucuronosyltransferase (ugt1a1), which is in accordance with the metabolism of retene as described by Huang et al. (2017). ...
Polycyclic aromatic hydrocarbons (PAHs) are widely spread environmental contaminants which affect developing organisms. It is known that improper activation of the aryl hydrocarbon receptor (AhR) by some PAHs contributes to toxicity, while other PAHs can disrupt cellular membrane function. The exact downstream mechanisms of AhR activation remain unresolved, especially with regard to cardiotoxicity. By exposing newly hatched rainbow trout alevins (Oncorhynchus mykiss) semi-statically to retene (32 µg l⁻¹; AhR agonist), fluoranthene (50 µg l⁻¹; weak AhR agonist and CYP1a inhibitor) and their binary mixture for 1, 3, 7 and 14 days, we aimed to uncover novel mechanisms of cardiotoxicity using a targeted microarray approach. At the end of the exposure, standard length, yolk area, blue sac disease (BSD) index and PAH body burden were measured, while the hearts were prepared for microarray analysis. Each exposure produced a unique toxicity profile. We observed that retene and the mixture, but not fluoranthene, significantly reduced growth by Day 14 compared to the control, while exposure to the mixture increased the BSD-index significantly from Day 3 onward. Body burden profiles were PAH-specific and correlated well with the exposure-specific upregulations of genes encoding for phase I and II enzymes. Exposure to the mixture over-represented pathways related to growth, amino acid and xenobiotic metabolism and oxidative stress responses. Alevins exposed to the individual PAHs displayed over-represented pathways involved in receptor signaling: retene downregulated genes with a role in G-protein signaling, while fluoranthene upregulated those involved in GABA signaling. Furthermore, exposure to retene and fluoranthene altered the expression of genes encoding for proteins involved in calcium- and potassium ion channels, which suggests affected heart structure and function. This study provides deeper understanding of the complexity of PAH toxicity and the necessity of investigating PAHs as mixtures and not as individual components.
